Defects within the insulin signaling pathways may lead to insulin resistance in peripheral tissues. The prevalence of obesity generally increases with age and is associated with insulin resistance characterized by an elevation in hepatic glucose production, impaired glucose tolerance and defects in the ability of the insulin receptor to transduce insulin signals. Cross-sectional studies have associated obesity and other components of the so-called metabolic syndrome with low-grade inflammation. A strong positive correlation has been reported between adipose tissue levels of adipocytokines (e.g., interleukin (IL)-6, tumor necrosis factor (TNF)) and the increased secretion of acute phase proteins by the liver of obese subjects. Hence, better understanding of the complex interplay between proinflammatory cytokines and insulin signal transduction could lead to the development of effective strategies to reduce or delay the progression of complications associated with insulin resistance states, such as Type 2 diabetes. We are in the process of completing a study whereby the effect of the thiol antioxidant pyrrolidine dithiocarbamate (PDTC) on IL-6 signaling has been elucidated. Our findings indicated that PDTC blocks IL-6?stimulated JAK/STAT3 signaling while restoring insulin responsiveness both in human HepG2 hepatoma and a primary culture of rat hepatocytes. A reduction in IL-6-mediated association of STAT3 with transcriptional coactivators was noted. Chromatin immunoprecipitation experiments are underway to validate these findings. We have just completed microarray and Northern blot analyses in HepG2 cells and found that PDTC suppresses IL-6?dependent induction of a number of genes, including those encoding acute-phase proteins. Taken together, these observations indicate a potential role for PDTC as a therapeutic agent in enhancing insulin sensitivity. It remains unknown whether PDTC can inhibit the development of insulin resistance in obesity and in aging, two chronic in vivo models of low grade inflammation. Therefore, we are planning an in vivo study to explore the role of PDTC in the liver in animal models of obesity and insulin resistance. High fat diet and diabetes increase oxidative injury including a proinflammatory response and lipid peroxidation leading to the development of non-alcoholic steatohepatitis (NASH). We will assess whether diabetes-prone mice maintained on high fatty diet and subjected to PDTC treatment will develop a proinflammatory response (e.g., acute-phase protein secretion) and ultimately lipid peroxidation in the liver will be evaluated. Similarly, it will be important to determine if this lipid dysregulation is STAT3-driven. A number of studies have indicated that PDTC is well tolerated in vivo. It is our hope that PDTC will be a useful tool and provide therapeutic/functional benefits by delaying onset and/or reducing duration of hepatic insulin resistance. In a second project, we investigated the role of the actin-binding protein filamin A (FLNa) in the modulation of proinflammatory cytokine signaling. In our previous study, FLNa was found to associate with the insulin receptor and attenuate insulin-induced activation of the Ras/MAPK pathway. Using the human melanoma cell line M2, which does not express FLNa, and its subclone A7, transfected with human FLNa cDNA, we observed that TNFalpha activation of NF-kappaB was greatly reduced in filamin-lacking cells, while insulin stimulation of the Ras/ERK cascade was significantly higher in these M2 cells. The coordinate induction of insulin signaling and repression of TNFalpha-mediated responses provides a molecular rationale for the antagonism between insulin and proinflammatory cytokines, and highlights the need to further investigate the role of FLNa as regulator of cytokine and insulin signaling. Hepatocytes respond to IL-6?mediated STAT3 activation with the synthesis and secretion of acute-phase proteins. In a series of recent experiments, we have successfully used RNA interference (RNAi) to knock down the expression of FLNa in HepG2 cells. The use of RNAi against FLNa mRNA will allow us to investigate the hypothesis that FLNa affects basal and IL-6-mediated activation of STAT3, as well as the induction of the IKK/NF-kB pathway in response to TNFalpha. Cell proliferation, survival, and expression of acute-phase protein genes and its correlation with insulin sensitivity with regard to glucose and lipid homeostasis will be explored in HepG2 hepatoma cell line. Within the next year, we are planning to prepare primary hepatic cells in culture from mice lacking FLNa to determine if the RNAi results in HepG2 cells parallel those of the primary hepatocytes. A mouse embryonic stem (ES) cell clone is available to us for the purpose of generating a FLNa gene knockout mouse. An increase in our understanding of the molecular mechanisms by which FLNa regulates cytokine and insulin signaling might allow identification of novel molecular targets for the treatment of low-grade proinflammatory diseases, such as obesity-linked insulin resistance. We are also investigating the hypothesis that FLNa affects STAT3 nuclear translocation and transcriptional activity to affect a diverse array of biological responses, including cell migration, inflammation and proliferation. STAT3 interaction with nuclear coactivators is known to be facilitated by phosphorylation at the Ser727 position. A marked elevation in STAT3 phosphorylation (Ser727) was observed in unstimulated FLNa-deficient M2 cells, but not in FLNa-containing A7 cells, and is reflected in high basal levels of ERK activity. Pharmacological inhibition of ERK with U0126 compound significantly decreased STAT3 Ser727 phosphorylation levels. Very little tyrosine phosphorylation of STAT3, if any, was observed in untreated M2 and A7 cells, suggesting that depletion of FLNa triggers constitutive serine phosphorylation of STAT3 partly through ERK activation. Immunofluorescence studies, using confocal microscopy, provided evidence that STAT3 was extensively localized in the nucleus of M2 cells. Indeed, reporter assays demonstrated constitutive STAT3-mediated transcriptional activation in M2 cells, but not in A7 cells, and did not respond to IL-6 with increased activity. We found that elevated basal levels of STAT3 activity was associated with greater invasiveness of the M2 cells that lack FLNa through higher expression and enzymatic activity of selective matrix metalloproteinases (MMPs). We are currently planning to assess the presence of a high affinity STAT3 binding element in relevant MMP promoters and determine whether STAT3 protein can bind directly to these promoters. Taken together, our investigation of the possible functional roles of FLNa in STAT3 signaling has now indicated that the level of serine phosphorylated STAT3 and metastasis could be inversely correlated to that of FLNa in melanoma.